Francisella tularensis can cause an acute, febrile, and sometimes fatal illness called tularemia. It is on the CDC's list of Category A agents due to its documented use for bioweapons, and there is not currently a licensed vaccine to protect against such use. Development of a live, attenuated vaccine strain would require non-antibiotic selectable markers for genetic manipulation of F. tularensis. The objective of this project is to develop a marker that can be used for this purpose, as well as for broader studies of this organism's physiology and metabolism in the future. The marker proposed here will be the basis for selection of a "pseudo-autotrophic" phenotype of F. tularensis. Our hypothesis is that metabolic manipulation of the pentose phosphate pathway, by introduction of the Calvin Cycle enzyme phosphoribulokinase (PRK), can lead to a casamino acid (CAA)-dependent phenotype that can be rescued by a second key enzyme from the Calvin Cycle, ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO). Two specific aims will enable us to test this hypothesis: [unreadable] 1. We will develop a stable PRK-positive strain of F. tularensis. [unreadable] 2. We will demonstrate the ability of RubisCO, in cis or in trans, to confer CAA-free growth through its normal function of carbon fixation. To fulfill these aims, we will insert a gene encoding bacterial PRK into a neutral site on the chromosome, under control of the F. tularensis groE promoter. Our unique approach to this will exclude the antibiotic marker used for construction of the strain. Plasmid-borne bacterial RubisCO, expressed from its own promoter, will be used to initially test the selection condition: restoration of growth on glucose minimal medium in the absence of CAA ("pseudoautotrophy"). Then, to demonstrate feasibility of RubisCO selection for vaccine strain development, it will be used in place of an antibiotic resistance marker to duplicate a previously described protocol for functional deletion of mglA, a gene known to play a role in virulence. Our expectation is that these experiments will lead to a valuable tool that can be used to develop a live, attenuated vaccine for tularemia. [unreadable] [unreadable] [unreadable]